Methods of isolation, expansion and differentiation of fetal stem cells from chorionic villus, amniotic fluid, and placenta and therapeutic uses thereof

ABSTRACT

The present invention is directed to pluripotent fetal stem cells derived from chorionic villus, amniotic fluid, and placenta and the methods for isolating, expanding and differentiating these cells, and their therapeutic uses such as manipulating the fetal stem cells by gene transfection and other means for therapeutic applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Phase Entry Under 35 U.S.C. §371of International Application No. PCT/US02/36966, filed Nov. 15, 2002which designated the U.S. and which claims benefit of U.S. ProvisionalApplication Ser. No. 60/335,878 filed Nov. 15, 2001 and U.S. ProvisionalApplication Ser. No. 60/356,295 filed Feb. 13, 2002.

FIELD OF THE INVENTION

This invention relates to the isolation, expansion and differentiationof fetal stem cells from chorionic villus, amniotic fluid, and placentaand therapeutic uses thereof.

BACKGROUND OF THE INVENTION

Stem cells are unique cell populations with the ability to undergo bothrenewal and differentiation. This fate choice is highly regulated byintrinsic signals and the external microenvironment. They can beidentified in many adult mammalian tissues, such as bone marrow,skeletal muscle, skin and adipose tissue, where they contribute toreplenishment of cells lost through normal cellular senescence orinjury. Although stem cells in adult tissues may be capable ofdeveloping into more cell types than originally thought, they have alimited cellular regeneration or turnover.

Stem cells have been reported to exist during embryonic development andpostnatally in bone marrow, skeletal muscle and skin. Embryonic stem(ES) cells are derived from the inner cell mass (ICM) at the blastulastage, and have the property of participating as totipotent cells whenplaced into host blastocysts. They are able not only to activate theexpression of genes restricted to each of the three embryonic germ (EG)layers, but they are also able to express receptors for a number ofdifferent soluble growth factors with established effects ondevelopmental pathways in vivo.

Adult stem cells, on the other hand, do not differentiate spontaneously,but can be induced to differentiate by applying appropriate growthconditions. Adult stem cells seem to be easier to maintain in culturethan ES cells. Adult stem cells have the disadvantage of not beingimmortal, and most of them lose their pluripotency after a definednumber of passages in culture. This short life-span may be a problem forclinical applications where a large amount of cells are needed.

In contrast to adult stem cells, ES cells, derived from blastocyst-stageearly mammalian embryos, have the ability to give rise to cells that notonly proliferate and replace themselves indefinitely, but that have thepotential to form any cell type. ES cells tend to differentiatespontaneously into various types of tissues; however, specific growthinduction conditions do not direct differentiation exclusively tospecific cell types. Two reports describing the isolation, long-termculture, and differentiation of such cells have generated tremendousexcitement in this regard and are herein incorporated by reference(Shamblott, Michael J., et al., “Derivation of Pluripotent Stem Cellsfrom Cultured Human Primordial Germ Cells,” Proc. Natl. Acad. Sci. USA,Vol. 95, pp. 13726-31, November 1998; Thomson, James A., et al.,“Embryonic Stem Cell Lines Derived from Human Blastocysts,” Science,Vol. 282, pp. 1145-47, Nov. 6, 1998). Although there is a greatscientific interest in ES cell research, the destruction of embryos inorder to harvest and experiment on ES cells still create unresolvedethical concerns.

Fetal tissue has been used in the past for autograft and allografttransplantation and tissue engineering research because of itspluripotency, proliferative ability and lack of immunogenecity. Fetalcells maintain a higher capacity to proliferate than adult cells and maypreserve their pluripotency longer in culture. However, fetal celltransplants are plagued by problems that are very difficult to overcome.Fetal tissue can be currently obtained from a biopsy of the fetus itselfduring gestation or from cord blood at birth; however, both proceduresare associated with a defined morbidity. Fetal tissue can also beobtained from aborted embryos, but this resource is limited. Beyond theethical concerns regarding the use of cells from aborted fetuses orliving fetuses, there are other issues which remain a challenge. Forexample, studies have shown that it generally takes about six fetuses toprovide enough material to treat one patient with Parkinson's disease.

Because stem cells, particularly pluripotent stem cells appear to be anexcellent resource for therapeutic applications, there is a great needfor a source of stem cells that is plentiful, easy to manipulate, andavoids ethical considerations.

SUMMARY OF THE INVENTION

We have discovered that chorionic villus, amniotic fluid, and placentaprovide an excellent source of pluripotent fetal stem cells fortherapeutic applications. These fetal stem cells have a better potentialfor expansion than adult stem cells and avoid the current controversiesassociated with the use of human embryonic stem cells. The c-kit^(pos)cells isolated from the chorionic villus, amniotic fluid and placentasamples differentiate into specific cell lineages, they do not needfeeder layers to grow, and most importantly, the isolation of thesecells does not require the sacrifice of human embryos for theirisolation, thus avoiding the current controversies associated with theuse of human embryonic stem cells.

Therefore, the present invention is directed to pluripotent fetal stemcells derived from chorionic villus, amniotic fluid, and placenta andthe methods for isolating, expanding and differentiating these cells,and their therapeutic uses such as manipulating the fetal stem cells bygene transfection and other means for therapeutic applications,including but not limited to enzyme replacement and gene therapy, tissueregeneration and replacement, including, for example burn and wounddressings.

In one aspect, the present invention provides a method for obtainingpluripotent human fetal stem cells comprising obtaining a chorionicvillus and/or amniotic fluid and/or placenta sample from a human subjectand isolating c-kit positive cells from the sample. The inventionfurther provides culturing or expanding the c-kit positive in a culturemedia before or after isolation. The chorionic villus, amniotic fluid orplacenta sample may be cryopreserved before isolating or differentiatingthe c-kit positive cells. Alternatively, the c-kit positive cells areisolated from the sample and then cryopreserved. The cells may becryopreserved before or after differentiation.

In yet another aspect, the present invention provides a method fordifferentiating the isolated pluripotent human fetal stem cells derivedfrom chorionic villus and/or amniotic fluid and/or placenta to cells ofdifferent lineages, including, but not limited to, osteogenic,adipogenic, myogenic, neurogenic, hematopoitic and endothelial lineages.Differentiation can be evidenced by, for example, changes in cellularmorphology and gene expression.

In a further aspect, the present invention provides a method fordifferentiating c-kit positive fetal stem cells contained within achorionic villus sample, amniotic fluid sample or a placenta sample tocells of different lineages, including, but not limited to, osteogenic,adipogenic, myogenic, neurogenic, hematopoietic, hepatic and endotheliallineages. The method comprises exposing the sample to one or moredifferentiation-inducing agents either in vivo or in vitro. Cells may beisolated from the sample before differentiation.

In yet another aspect, the present invention provides a method forassessing viability, proliferation potential, and longevity of thepluripotent human fetal stem cells derived from chorionic villus,amniotic fluid and placenta.

In another aspect the invention provides a method of treating disease ina human comprising administering to a human in need thereof asubstantially enriched population of cells comprising pluripotent c-kitpositive human fetal stem cells which have been differentiated to alineage selected from osteogenic, hematopoietic, adipogenic, myogenic,hepatic, neurogenic and endothelial cell lineage. For example,Parkinson's disease can be treated with the isolated pluripotent c-kitpositive stem cells of the present invention either directly, or afterdifferentiating such cells into a neuronal cell lineage capable ofproducing dopamine.

The invention further provides a method of transplanting into a human inneed thereof a substantially enriched population of cells comprisingpluripotent c-kit positive human fetal stem cells which have beendifferentiated to a lineage selected from osteogenic, hematopoietic,adipogenic, myogenic, hepatic, neurogenic and endothelial phenotype.

In another aspect, the invention provides a composition suitable forbonemarrow transplantation comprising a substantially enrichedpopulation of cells comprising pluripotent c-kit positive human fetalstem cells which have been differentiated to a lineage selected fromosteogenic, hematopoietic, adipogenic, myogenic, hepatic, neurogenic andendothelial phenotype.

Further, the invention provides a method of obtaining a population ofcells enriched for pluripotent fetal stem cells, comprising isolating atissue specimen from the chorionic villus of a human placenta.

The invention also provides a method of obtaining a population of cellsenriched for pluripotent fetal stem cells, comprising isolating a tissuespecimen containing said cells from human placenta, chorionic villus oramniotic fluid.

In yet another aspect the invention provides a method of obtaining apopulation of cells enriched for pluripotent fetal stem cells,comprising selecting c-kit positive cells from placenta.

The invention further provides a method of obtaining a population ofcells enriched for pluripotent fetal stem cells, comprising the steps ofcryopreseverving a tissue specimen from the chorionic villus, amnioticfluid or placenta, and thawing the cryopreserved specimen at a laterdate and selecting c-kit positive cells.

In another aspect, the invention provides a method of producing apopulation of cells enriched for pluripotent fetal stem cells comprisingisolating c-kit positive cells from the chorionic villus, placenta oramniotic fluid, and proliferating the cells in culture medium.

In another aspect, the invention provides a method of producingdifferentiated tissue comprising providing a tissue specimen fromchorionic villus, amniotic fluid or placenta, culturing the tissue underconditions that cause c-kit positive cells to proliferate; and uponinduction cause the c-kit positive cells to differentiate.

The invention also provides a method of the invention provides a methodof obtaining a population of cells enriched for pluripotent fetal stemcells, comprising isolating a tissue specimen from the chorionic villusof a human placenta, placenta, or amniotic fluid further comprisingusing negative selection to enrich c-kit positive cells from thechorionic villus.

In yeat another aspect, the invention provides a method of obtaining apopulation of cells enriched for pluripotent fetal stem cells,comprising isolating a tissue specimen containing said cells from humanplacenta, chorionic villus or amniotic fluid wherein the cells aresubsequently cryopreserved.

Finally, the present invention provides therapeutic applications for thefetal stem cells derived from a chorionic villus and/or an amnioticfluid and/or a placenta sample including, but not limited to (a)autologous/heterologous enzyme replacement therapy; (b)autologous/heterologous transgene carriers in gene therapy; (c)autologous/heterologous tissue regeneration/replacement therapy; (d)reconstructive treatment by surgical implantation; (e) reconstructivetreatment of tissues with products of these cells; and (f) tissueengineering.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1G show results from chorionic villi and amniotic cellcharacterization experiments. Between 0.8 and 3% of the amniotic andchorionic villi cells were c-kit^(pos) [1A]. The c-kit^(pos) cells didnot stain with mouse stage specific embryonic antigen 1 [1B], butstained positively for human stage specific embryonic antigens 3 and 4[1C and 1D]. Analyses of late passage c-kit^(pos) cells (PD 200) showeda normal karyotype [1E]. Telomerase activity was evaluated using theTelomerase Repeat Amplification Protocol (TRAP) assay [1F]. Thechorionic villi and amniotic c-kit^(pos) cells were telomerase positive(lane 1). Upon differentiation into specific lineages, telomeraseactivity diminished to undetectable levels (Lane 2). Lane 3 shows thepositive control. Lane 4 represents negative control cell lysate,showing no telomerase activity. The telomeric length was evaluated byterminal restriction fragment (TRF) measurement [1G]. C-kit^(pos) cellshad similar telomere lengths, both at early and late passages (250 PD)(lane 3 and 4, respectively) as compared with a high molecular weightmarker, approximately 10.2 kbp (lane 2). Lane 1 represents a lowmolecular weight marker.

FIGS. 2A-2L demonstrate osteogenic induction of the c-kit^(pos) cellsisolated from chorionic villi and amniotic fluid. The shape of chorionicvilli and amniotic c-kit^(pos) cells treated with osteogenic-inducingmedium after 4 days of induction changed to an osteoblast-likeappearance [2A], whereas cells in the control medium did not lose theirspindle-shaped phenotype [2B]. Alkaline phosphatase activity wasquantified in c-kit^(pos) cells that were incubated withosteogenic-inducing and control medium for 32 days [2C]. Numbersrepresent alkaline phosphatase production in nMol p-Nitrophenyl/min/10⁶cells, showing a peak of production at day 16 (solid line); whereasc-kit^(pos) cells grown in control medium (shaded line) or c-kit^(neg)cells grown in osteogenic conditions (dotted line) did not show anyalkaline phosphatase production. C-kit^(pos) cells treated withosteogenic-inducing medium and with control medium stained for alkalinephosphatase after 4, 8, 16, 24 and 32 days [2D]. Strong alkalinephosphatase staining was noted in the osteogenic-induced cells startingat day 16, and remained high thereafter. C-kit^(pos) cells grown incontrol medium did not show any alkaline phosphatase staining. Whenconfluent, the cells formed typical lamellar structures similar to thosefound in bone [2F]. C-kit^(pos) cells in control medium did not form anylamellar structures [2E]. Mineralization of cells was quantified using achemical assay for calcium [2G]. Numbers represent calcium deposition inmg/dl. Osteogenic-induced ckit^(pos) cells showed a significant increaseof calcium deposition starting at day 16 (solid line). No calciumdeposition was detected in ckit^(pos) cells grown in control medium(shaded line) or ckit^(pos) cells grown in osteogenic conditions (dottedline). Furthermore cells treated with control medium or withosteogenic-inducing medium were analyzed using von Kossa staining after32 days in culture (40×). The osteogenic-induced cells showedsignificant mineralization starting at day 16 [2H]. No mineralizationoccurred at any time point in cells grown in control medium [2I]. RNAwas isolated from amniotic c-kit^(pos) cells grown in control medium(lanes 1, 2, 3 and 4) and osteogenic-inducing medium (lanes 5, 6, 7 and8). RT-PCR was performed using primers for alkaline phosphatase, cbfa1,osteocalcin and β2-microglobulin at days 8, 16, 24 and 32 [2G]. RT-PCRshowed upregulation of cbfa1 and osteocalcin at day 8 and it confirmedthe upregulation of alkaline phosphatase in the osteogenic-induced cells[2J]. C-kit^(pos) cells were seeded on hydroxyapatite-collagenscaffolds, induced into an osteogenic lineage, implanted subcutaneouslyin athymic mice, and harvested after 4 and 8 weeks. Bone-like tissue wasevident, surrounded by an extracellular matrix. Toluidine blue stainingconfirmed the osteogenic phenotype. Large calcified areas within theimplanted tissue stained positively with von Kossa, indicating boneformation [2K]. Non seeded scaffold were implanted and used as control[2L].

FIGS. 3A-3F demonstrate adipogenic induction of the c-kit^(pos) cellsisolated from chorionic villi and amniotic fluid. Clusters of adipocytesappeared at 8 days [3A], and the percentage of cells increased with timeuntil Oil-O-Red was uniformly staining the adipogenesis-induced cells atday 16 [3B]. C-kit^(pos) cells cultured in control medium did not showany lipid deposits at day 16 [3C]. RNA was isolated from c-kit^(pos)cells grown in control (lanes 1 and 2) and adipogenic-inducing (lanes 3and 4) medium [3D]. RT-PCR was performed using primers for PPARγ2,lipoprotein lipase and β2-microglobulin at days 8 and 16, as indicated.Upregulation of PPARγ2 and lipoprotein lipase in cells grown inadipogenic-inducing medium was noted at days 8 and 16 (lanes 3 and 4).C-kit^(pos) cells were seeded on polyglycolic acid polymer scaffolds.Cells were induced into an adipogenic lineage. The scaffolds wereimplanted subcutaneously in athymic mice, harvested after 4 and 8 weeksand analyzed. The retrieved scaffolds showed the formation of fattytissues grossly [3E]. The presence of adipose tissue was confirmed withOil-O-Red staining (200× magnification) [3F].

FIGS. 4A-4I demonstrate myogenic induction of the c-kit^(pos) cellsisolated from chorionic villi and amniotic fluid. Under myogenicconditions the c-kit^(pos) cells fused into multinucleated cells at day4 [4A] and formed myotube-like structures after 8 days [4B].Multinucleated cells stained green for sarcomeric tropomyosin [4C] anddesmin [4D] expression 16 days after myogenic induction. Cell nucleiwere stained blue using DAPI. Untreated cells did not stain forsarcomeric tropomyosin [4E] or desmin [4F]. RNA was isolated fromc-kit^(pos) cells grown in control (lanes 1 and 2) and myogenic-inducing(lanes 3 and 4) medium [4G]. RT-PCR was performed using primers forMyoD, MRF4 (herculin, Myf6), and desmin at days 8 and 16.Myogenic-induced cells showed a strong upregulation of desmin expressionat day 16 (lane 4). MyoD and MRF4 were induced with myogenic treatmentat day 8 (lane 1). Specific PCR amplified DNA fragments of MyoD, MRF4and Desmin could not be detected in the control cells at days 8 and 16(lanes 1 and 2). C-kit^(pos) cells were labeled with the fluorescencemarker PKH26 and were induced into a myogenic lineage. The myogeniccells were injected into the hindlimb musculature of athymic mice andwere retrieved after 4 weeks. The injected myogenic cells showed theformation of muscle tissue (m) which expressed desmin [4H] andmaintained its fluorescence [4I]. The native muscle (n) did not expressany fluorescence.

FIGS. 5A-5F demonstrate endothelial induction of the c-kit^(pos) cellsisolated from chorionic villi and amniotic fluid. Ckit^(pos) cells werecultured as monolayers in PBS-gelatin coated dishes with EBM-2 and bFGFand showed a typical endothelial appearance in vitro [5A]. The fullydifferentiated endothelial cells stained for the endothelial specificmarkers FVIII [5B], KDR [5C] and P1H12 [5D]. Once cultured in matrigelthe cells were able to form capillary structures over time [5E]. Inorder to confirm the phenotypic changes we performed RT-PCR 5[F]. CD31and VCAM showed a marked increased in the ckit^(pos) cells induced inendothelial medium (lane 2). Ckit^(pos) cells cultured in control medium(lane 1) did not show any gene amplification.

FIGS. 6A-6E demonstrate neurogenic induction of the c-kit^(pos) cellsisolated from chorionic villi and amniotic fluid. Ckit^(pos) cellscultured under neurogenic inducing conditions changed their morphologywithin the first 24 hours. The cell cytoplasm retracted towards thenucleus, forming contracted multipolar structures, with primary andsecondary branches, and cone-like terminal expansions [6A]. Thedifferentiated cells stained for specific neurogenic markers β IIITubulin [6B], Nestin [6C], and glial fibrillary acidic protein (GFAP)[6D]. Only the C-kit^(pos) cells cultured under neurogenic conditionsshowed the secretion of glutamic acid in the collected medium.Furthermore the secretion of glutamic acid could be induced (KCl; 20 minin 50 mM KCl buffer) [6E].

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon a discovery that chorionic villus,amniotic fluid, and placenta cells can be used to obtain a population ofstem cells which are comparable to embryonic stem cells in theirpluripotent differentiation capacity and therefore are a viable sourceof stem cells that can be used therapeutically.

Chorionic villus sampling and amniocentesis are well establishedtechniques for the collection of tissue from the human embryo (10 to 12weeks) and for the collection of fluid from the human fetus (12 weeks toterm), respectively. Chorionic villus sampling is performed on pregnantmammal, preferably human, and has been in use since the 1980s. Thisprocedure involves taking a sample of the chorion frondosum—that part ofthe chorionic membrane containing the villi. The chorionic membrane isthe outer sac which surrounds the developing fetus. Chorionic villi aremicroscopic, finger-like projections that emerge from the chorionicmembrane and eventually form the placenta. The cells that make up thechorionic villi are of fetal origin.

In humans, chorionic villus sampling is best performed between 10 and 12weeks of pregnancy. The procedure is performed either through the vaginaand the cervix (transcervically) or through the abdomen(transabdominally) depending upon the preferences of the patient or thedoctor. In some cases, the location of the placenta dictates whichmethod the doctor uses. For the transcervical procedure, the woman lieson an examining table on her back with her feet in stirrups. The woman'svaginal area is thoroughly cleansed with an antiseptic, a sterilespeculum is inserted into her vagina and opened, and the cervix iscleansed with an antiseptic. Using ultrasound (a device which uses soundwaves to visualize internal organs) as a guide, the doctor inserts athin, plastic tube called a catheter through the cervix and into theuterus. The passage of the catheter through the cervix may causecramping. The doctor carefully watches the image produced by theultrasound and advances the catheter to the chorionic villi. By applyingsuction from the syringe attached to the other end of the catheter, asmall sample of the chorionic villi are obtained. A cramping or pinchingfeeling may be felt as the sample is being taken. The catheter is theneasily withdrawn.

For the transabdominal method, the woman lies on her back on anexamining table. Ultrasound enables the doctor to locate the placenta.The specific area on the woman's abdomen is cleansed thoroughly with anantiseptic and a local anesthetic may be injected to numb the area. Withultrasound guidance, a long needle is inserted through the woman'sabdominal wall, through the uterine wall and to the chorionic villi. Thesample is obtained by applying suction from the syringe. The chorionicvillus sample is immediately placed a into nutrient medium.

Amniotic fluid is obtained using amniocentesis. The word amniocentesisliterally means “puncture of the amnion,” the thin-walled sac of fluidin which a developing fetus is suspended during pregnancy. During thesampling procedure, the obstetrician inserts a very fine needle throughthe woman's abdomen into the uterus and amniotic sac and withdrawsapproximately one ounce of amniotic fluid.

The physician uses ultrasound images to guide needle placement andcollect the sample, thereby minimizing the risk of fetal injury and theneed for repeated needle insertions. Once the sample is collected, thewoman can return home after a brief observation period. She may beinstructed to rest for the first 24 hours and to avoid heavy lifting fortwo days. Consequently, the fetal cells contained in the fluid areisolated and grown as explained below.

These techniques may be used to obtain chorionic villus and amnioticfluid samples in accordance with the present invention. Cultured cellsfrom the chorionic villi or amniotic fluid of pregnancies have been usedwidely for the prenatal diagnosis of genetic disorders. The morphologicheterogeneity of these cells is well known. Numerous cell types from all3 germ layers are found in the placenta and the amniotic fluid atdifferent levels of differentiation (6). Large quantities of chorionicvilli and amniotic fluid are available during pregnancy and at the timeof birth, and cells can be easily obtained from these sources. The sameis true for placenta, which is obtainable after birth.

A sample of placenta may be obtained using a punch-biopsy, a scalpel orhomogenizing the placenta or a portion thereof using, for example, ablender. The homogenate may then be used as a source of cells.

Stem cell differentiation requires cell-cell contact andcell-extracellular matrix interactions. While not wishing to be bound bya particular theory, it is believed that chorionic villus, amnioticfluid and placenta make a good source of undifferentiated cells becausethe cells liberated in the chorionic villus and amniotic fluid from thefetus during development may not receive any signal of differentiation,and may be able to maintain their “pluripotential” state. We havediscovered that the preferred cells are c-kit positive. Thus, the c-kitmarker can be used to isolate these cells. As used herein the terms“pluripotent” or “pluripotential” cell refers to a cell that hascomplete differentiation versatility, i.e., the capacity todifferentiate into at least osteogenic phenotype, hematopoieticphenotype, adipogenic phenotype, myogenic phenotype, hepatic phenotypeand endothelial phenotype in appropriate inducing conditions, preferablythe pluripotent cell has the capacity to differentiate to any of themammalian body's about 260 different cell types.

The c-kit gene encodes a tyrosine kinase growth factor receptor for StemCell Factor (SCF), also called mast cell growth factor, that isessential for hematopoiesis, melanogenesis and fertility. The larger 45kDa form is processed to generate a 31 kDa soluble factor while thesmaller 32 kDa form gives rise to a 23 kDa factor. Expression of the twoalternatively spliced forms is somewhat tissue-specific; the 31 kDa formof SCF is expressed in fibro-blasts and thymus tissue while the 23 kDafactor is found in spleen, testis, placenta and cerebellum. The c-kitreceptor protein, also known as c-Kit receptor, Steel factor receptor,stem cell factor receptor and CD117 in standardized terminology ofleukocyte antigens, is constitutively expressed in hematopoietic stemcells, mast cells, germ cells, melanocytes, certain basal epithelialcells, luminal epithelium of breast, and the interstitial cells of Cajalof the gastrointestinal tract. The c-kit receptor plays a fundamentalrole during the establishment, maintenance and function of germ cells.In the embryonal gonad the c-kit receptor and its ligand SCF arerequired for the survival and proliferation of primordial germ cells. Inthe postnatal animal, c-kit/SCF are required for production of themature gametes in response to gonadotropic hormones, i.e. for thesurvival and/or proliferation of the only proliferating germ cells ofthe testis, the spermatogonia, and for the growth and maturation of theoocytes. Experiments have shown that c-kit is a potent growth factor forprimitive hematopoietic cell proliferation in vitro. In mice, loss ofeither SCF or c-kit due to mutations in their loci results in macrocyticanemia, leading to death in utero or within the first postnatal days.

Antibodies reactive with the c-kit or portions thereof can be used toisolate c-kit positive cells. In a preferred embodiment, the antibodiesspecifically bind with the c-kit or a portion thereof. The antibodiescan be polyclonal or monoclonal, and the term antibody is intended toencompass polyclonal and monoclonal antibodies, and functional fragmentsthereof. The terms polyclonal and monoclonal refer to the degree ofhomogeneity of an antibody preparation, and are not intended to belimited to particular methods of production.

Therefore, examples of antibodies useful according to the presentinvention include antibodies recognizing the c-kit. Such antibodies areherein referred to as “c-kit antibodies.” Examples of commerciallyavailable useful c-kit antibodies include, but are not limited toantibodies in table 1, that can be purchased from Santa CruzBiotechnology, Inc.

Antibody Cat.# Isotype Epitope Applications Species SCF (N-19) sc-1302goat IgG N-terminus WB, IP, IHC, human (h) ELISA c-Kit (C-19) sc-168rabbit IgG C-terminus WB, IP, IHC, mouse, rat, (h) PAR human c-Kit(M-14) sc-1494 goat IgG C-terminus WB, IP, IHC mouse, rat, (m) humanc-Kit (Ab 81) sc-13508 mouse IgG₁ FL (h) WB, IP, IHC human FCM c-Kit(C-14) sc-1493 goat IgG C-terminus WB, IP, IHC human> (h) mouse c-Kit(104D2) sc-19983 mouse IgG₁ n/a IHC, FCM human c-Kit (H-300) sc-5535rabbit IgG 23-322 (h) WB, IP, IHC mouse rat, human c-Kit (E-1) sc-17806mouse IgG₁ 23-322 (h) WB, IHC human

The preferred antibody is c-Kit (E-1), which is a mouse monoclonal igGrecognizing an epitope corresponding to amni acids 23-322 mapping nearthe c-kit N-temminys and recognizes both c-Kit of human origin by bothWestern blotting and immunihistochemistry.

Additional examples of commercially available antibodies include, butare not limited to YB5.B8 monoclonal antibody, specific for human CD117(eBioscience, San Diego, Calif.); an antibody produced against a humanleucaemic cell line UT7 transfected with CD117 cDNA (ChemiconInternational, Temecula, Calif.); a polyclonal antibody produced againstthe C-terminal end of CD117 (Assay Designs Inc., Ann Arbor, Mich.,catalog No. 90572); clone 28 c-kit monoclonal antibody (catalog no.612318, from BD Transduction Laboratories, Franklin Lakes, N.J.); ac-kit tyrosine kinase receptor antibody ab1462, which is a rabbitpolyclonal anti-human c-kit tyrosine kinase receptor antibody wasgenerated using a synthetic KLH-conjugated peptide corresponding to thecarboxy-terminus of the CD117; and a monoclonal 13CD anti c-kit antibodyfrom Zymed Laboratories Inc. (South San Francisco, Calif.).

Further, antibodies recognizing c-kit or fragments thereof may beobtained or prepared as discussed in U.S. Pat. No. 5,454,533,incorporated herein by reference. The c-kit antigen can be contactedwith an antibody, such as various c-kit monoclonal antibodies, whichhave specificity for the c-kit antigen. A c-kit antibody ischaracterized by binding to the c-kit protein or fragments thereof underWestern blot conditions from reducing SDS-PAGE gels. For example, theCD117 antigen of c-kit has a molecular weight, based on commerciallyavailable standards, in the range of about 145 kDa.

The terms “specific binding” or “specifically binding”, as used herein,refers to the interaction between a c-kit or a fragment thereofexpressed by a cell present in a chorionic villus, amniotic fluid orplacenta sample, and an antibody. The interaction is dependent upon thepresence of a particular structure, i.e., the antigenic determinant orepitope of c-kit, of the c-kit recognized by the binding molecule, i.e.the c-kit antibody. For example, if an antibody is specific for epitope“A” of c-kit, the presence of a protein containing epitope A (or free,unlabeled A) in a reaction containing labeled “A” and the antibody willreduce the amount of labeled A bound to the antibody.

Additionally, antibodies to c-kit antigen or fragments thereof can beobtained by immunizing a xenogeneic immunocompetent mammalian host(including murine, rodentia, lagomorpha, ovine, porcine, bovine, etc.)with human c-kit or fragments thereof expressing cells. The choice of aparticular host is primarily one of convenience. A suitable progenitorcell population for immunization can be obtained, for example byisolating c-kit positive cells from tissues or cell cultures.Immunizations are performed in accordance with conventional techniques,where the cells may be injected subcutaneously, intramuscularly,intraperitoneally, intravascularly, etc. Normally, from about 10⁶ to 10⁸cells will be used, which may be divided up into one or more injections,usually not more than about 8 injections, over a period of from aboutone to three weeks. The injections may be with or without adjuvant, e.g.complete or incomplete Freund's adjuvant, specol, alum, etc.

After completion of the immunization schedule, the antiserum may beharvested in accordance with conventional ways to provide polygonalantisera specific for the surface membrane proteins of progenitor cells,including the c-kit antigen or fragments thereof. Lymphocytes areharvested from the appropriate lymphoid tissue, e.g. spleen, draininglymph node, etc., and fused with an appropriate fusion partner, usuallya myeloma line, producing a hybridoma secreting a specific monoclonalantibody. Screening clones of hybridomas for the antigenic specificityof interest is performed in accordance with conventional methods.

Antibodies against c-kit or fragments thereof can be produced as asingle chain, instead of the normal multimeric structure. Single chainantibodies are described in Jost et al., 269 J. Biol. Chem. 26267-73(1994), incorporated herein by reference, and others. DNA sequencesencoding the variable region of the heavy chain and the variable regionof the light chain are ligated to a spacer encoding at least about 4amino acids of small neutral amino acids, including glycine or serine.The protein encoded by this fusion allows assembly of a functionalvariable region that retains the specificity and affinity of theoriginal antibody.

Antibodies against c-kit or fragments thereof can be produced by use ofIg cDNA for construction of chimeric immunoglobulin genes (Liu et al.,84 Proc. Natl. Acad. Sci. 3439 (1987) and 139 J. Immunol. 3521 (1987),incorporated herein by reference. mRNA is isolated from a hybridoma orother cell producing the antibody and used to produce cDNA. The cDNA ofinterest may be amplified by the polymerase chain reaction usingspecific primers (U.S. Pat. Nos. 4,683,195 and 4,683,202).Alternatively, a library is made and screened to isolate the sequence ofinterest. The DNA sequence encoding the variable region of the antibodyis then fused to human constant region sequences. The sequences of humanconstant regions genes may be found in Kabat et al., “Sequences ofProteins of Immunological Interest” N.I.H. publication No. 91-3242(1991). Human C region genes are readily available from known clones.The chimeric, humanized antibody is then expressed by conventionalmethods.

Antibodies against c-kit or fragments thereof can also be produced asantibody fragments, such as Fv, F(ab′)₂ and Fab. Antibody fragments maybe prepared by cleavage of the intact protein, e.g. by protease orchemical cleavage. Alternatively, a truncated gene is designed. Forexample, a chimeric gene encoding a portion of the F(ab′)₂ fragmentwould include DNA sequences encoding the CH1 domain and hinge region ofthe H chain, followed by a translational stop codon to yield thetruncated molecule.

The c-kit positive cell selection can be by any suitable means known inthe art, including flow cytometry, such as by fluorescence activatedcell sorting using a fluorochrome conjugated c-kit antibody. Theselection can also be by high gradient magnetic selection using c-kitantibody is conjugated to magnetic particles. Any other suitable methodincluding attachment to and disattachment from solid phase, is alsocontemplated within the scope of the invention.

One of skill in the art can derive the population of cells byimmunoselection using an c-kit antibody. The population of cells shouldcontain at least 30% c-kit positive (c-kit⁺ or c-kit^(pos)) pluripotentfetal stem cells, preferably at least 50-70% c-kit⁺ fetal stem cells,and more preferably greater than 90% c-kit⁺ fetal stem cells. Mostpreferable would be a substantially pure population of c-kit⁺ fetal stemcells, comprising at least 95% c-kit⁺ fetal stem cells.

The number of c-kit positive cells in a cell population can bedetermined in any well known method known to one skilled in the art. Forexample, FACS analysis can be used as shown in FIG. 1A. Alternatively,magnetic cell sorting technology (MACS) can be used to separate cells(see, e.g. Miltenyi Biotech, Inc., Auburn, Calif.). In MACS, the c-kitpositive cells can be separated from the mixture of chorionic villuscells, amniotic fluid, and placenta cells to very high purity. The c-kitpositive cells are specifically labeled with super-paramagnetic MACSMicroBeads which can be designed to bind to either the c-kit antigendirectly or to the antibody recognizing c-kit. After magnetic labeling,the cells are passed through a separation column which is placed in astrong permanent magnet. The column matrix serves to create ahigh-gradient magnetic field. The magnetically labeled cells areretained in the column while non-labeled cells pass through. Afterremoval of the column from the magnetic field, the magnetically retainedcells are eluted. Both labeled and non-labeled fractions can becompletely recovered.

The in vitro cell cultures described herein containing an enrichedpopulation of c-kit positive pluripotent fetal stem cells are generallycharacterized in that the cultures stain positive for c-kit and SSAE3and SSAE4, produce progeny cells that can differentiate into at leasttwo, preferably three, most preferably at least all of the followingcell lineages: osteogenic, adipogenic, neurogenic, myogenic,hematopoietic, hepatic and endothelial cell lineages in the presence ofdifferentiation-inducing conditions of which examples are described inthe Example below. Further examples of differentiation-inducing agenstand combinations thereof for differentiating desired cell lineages canbe found at Stem Cells: Scientific Progress and Future ResearchDirections. (Appendix D. Department of Health and Human Services. June2001. www.nih.gov/news/stemcell/scireport.htm)

Immunostaining. Biological samples including the cells isolated fromchrorionic villus samples, amniotic fluid samples or placenta areassayed for the presence of c-kit⁺ fetal stem cells by any convenientimmunoassay method for the presence of cells expressing the c-kit, boundby the c-kit antibodies. Assays may be performed on cell lysates, intactcells, frozen sections, etc.

Cell Sorting. The use of cell surface antigens to fetal stem cells, suchas c-kit provides a means for the positive immunoselection of fetal stemcell populations, as well as for the phenotypic analysis of progenitorcell populations using, for example, flow cytometry. Cells selected forexpression of c-kit antigen may be further purified by selection forother stem cell and progenitor cell markers, including, but not limitedto SSAE3 and SSAE4 human embryonic stem stage specific markers.

Alternatively, for the preparation of substantially pure pluripotentfetal stem cells, a subset of stem cells can be separated from othercells on the basis of c-kit antibody binding and the c-kit positivefetal stem cells may be further separated by binding to other surfacemarkers known in the art.

Procedures for separation may include magnetic separation, usingantibody-coated magnetic beads, affinity chromatography and “panning”with antibody attached to a solid matrix, e.g. plate, or otherconvenient technique. Techniques providing accurate separation includefluorescence activated cell sorters, which can have varying degrees ofsophistication, such as multiple color channels, low angle and obtuselight scattering detecting channels, impedance channels, etc. Dead cellsmay be eliminated by selection with dyes associated with dead cells(propidium iodide (PI), LDS). Any technique may be employed which is notunduly detrimental to the viability of the selected cells.

Conveniently, the antibodies are conjugated with labels to allow forease of separation of the particular cell type, e.g. magnetic beads;biotin, which binds with high affinity to avidin or streptavidin;fluorochromes, which can be used with a fluorescence activated cellsorter; haptens; and the like. Multi-color analyses may be employed withthe FACS or in a combination of immunomagnetic separation and flowcytometry. Multi-color analysis is of interest for the separation ofcells based on multiple surface antigens, e.g. c-kit⁺, and antibodiesrecognizing SSAE3 and SSAE4 cell markers. Fluorochromes which find usein a multi-color analysis include phycobiliproteins, e.g. phycoeryirinand allophycocyanins; fluorescein and Texas red. A negative designationindicates that the level of staining is at or below the brightness of anisotype matched negative control. A dim designation indicates that thelevel of staining may be near the level of a negative stain, but mayalso be brighter than an isotype matched control.

In one embodiment, the c-kit antibody is directly or indirectlyconjugated to a magnetic reagent, such as a superparamagneticmicroparticle (microparticle). Direct conjugation to a magnetic particleis achieved by use of various chemical linking groups, as known in theart. Antibody can be coupled to the microparticles through side chainamino or sufhydryl groups and heterofunctional cross-linking reagents. Alarge number of heterofunctional compounds are available for linking toentities. A preferred linking group is 3-(2-pyridyidithio)propionic acidN-hydroxysuccinimide ester (SPDP) or4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid N-hydroxysuccinimideester (SMCC) with a reactive sulfhydryl group on the antibody and areactive amino group on the magnetic particle.

Alternatively, c-kit antibody is indirectly coupled to the magneticparticles. The antibody is directly conjugated to a hapten, andhapten-specific, second stage antibodies are conjugated to theparticles. Suitable haptens include digoxin, digoxigenin, FITC,dinitrophenyl, nitrophenyl, avidin, biotin, etc. Methods for conjugationof the hapten to a protein, i.e. are known in the art, and kits for suchconjugations are commercially available.

To practice the method, the c-kit antibody (Ab) is added to a cellsample. The amount of c-kit Ab necessary to bind a particular cellsubset is empirically determined by performing a test separation andanalysis. The cells and c-kit antibody are incubated for a period oftime sufficient for complexes to form, usually at least about 5 min,more usually at least about 10 min, and usually not more than one hr,more usually not more than about 30 min.

The cells may additionally be incubated with antibodies or bindingmolecules specific for cell surface markers known to be present orabsent on the fetal stem cells. For example, cells expressing SSAE1marker can be negatively selected for.

The labeled cells are separated in accordance with the specific antibodypreparation. Fluorochrome labeled antibodies are useful for FACSseparation, magnetic particles for immunomagnetic selection,particularly high gradient magnetic selection (HGMS), etc. Exemplarymagnetic separation devices are described in WO 90/07380,PCT/US96/00953, and EP 438,520.

The purified cell population may be collected in any appropriate medium.Various media are commercially available and may be used, includingDulbecco's Modified Eagle Medium (DMEM), Hank's Basic Salt Solution(HBSS), Dulbecco's phosphate buffered saline (dPBS), RPMI, Iscove'smodified Dulbecco's medium (IMDM), phosphate buffered saline (PBS) with5 mM EDTA, etc., frequently supplemented with fetal calf serum (FCS),bovine serum albumin (BSA), human serum albumin (HSA), etc. Preferredculture media include DMEM, F-12, M1 99, RPMI.

Populations highly enriched for pluripotent fetal stem cells areachieved in this manner. The desired cells will be 30% or more of thecell composition, preferably 50% or more of the cell population, morepreferably 90% or more of the cell population, and most preferably 95%or more (substantially pure) of the cell population.

The use of substantially purified or enriched c-kit positive pluripotentfetal stem cells of the present invention are useful in a variety ofways. The c-kit positive cells can be used to reconstitute a host whosecells have been lost through disease or injury. Genetic diseasesassociated with cells may be treated by genetic modification ofautologous or allogeneic stem cells to correct a genetic defect or treatto protect against disease.

Alternatively, normal allogeneic fetal stem cells may be transplanted.Diseases other than those associated with cells may also be treated,where the disease is related to the lack of a particular secretedproduct such as hormone, enzyme, growth factor, or the like. CNSdisorders encompass numerous afflictions such as neurodegenerativediseases (e.g. Alzheimer's and Parkinson's), acute brain injury (e.g.stroke, head injury, cerebral palsy) and a large number of CNSdysfunctions (e.g. depression, epilepsy, and schizophrenia). In recentyears neurodegenerative disease has become an important concern due tothe expanding elderly population which is at greatest risk for thesedisorders. These diseases, which include Alzheimer's Disease, MultipleSclerosis (MS), Huntington's Disease, Amyotrophic Lateral Sclerosis, andParkinson's Disease, have been linked to the degeneration of neuralcells in particular locations of the CNS, leading to the inability ofthese cells or the brain region to carry out their intended function. Byproviding for maturation, proliferation and differentiation into one ormore selected lineages through specific different growth factors theprogenitor cells may be used as a source of committed cells. Thepluripotent fetal stem cells according to the present invention can alsobe used to produce a variety of blood cell types, including myeloid andlymphoid cells, as well as early hematopoietic cells (see, Bjornson etal., 283 Science 534 (1999), incorporated herein by reference).

A variety of cell differentiation inducing agents can be use todifferentiate the pluripotent fetal stem cells of the present inventioninto different phenotypes. To determine the differentiation status ofthe stem cells, the phenotypic characteristic of the cells are observedusing conventional methods such as light microscopy to detect cellmorphology (see, e.g., FIGS. 2-6), RT-PCT to detect cell lineagespecific transcription, and immunocytochemistry to detect cell surfacemarkers specifically expressed in a particulate cell lineage. Forexample, genes expressed during the osteogenic differentiation serve asmarkers of the stem cells differentiating into osteogenic lineage (Long,Blood Cells Mol Dis 2001 May-June; 27(3):677-90).

The c-kit positive fetal stem cells may also be used in the isolationand evaluation of factors associated with the differentiation andmaturation of cells. Thus, the cells may be used in assays to determinethe activity of media, such as conditioned media, evaluate fluids forgrowth factor activity, involvement with dedication of lineages, or thelike.

The isolated c-kit positive fetal stem cells may be cryopreserved, i.e.frozen at liquid nitrogen temperatures and stored for long periods oftime, being thawed and capable of being reused. The cells will usuallybe stored in 5% DMSO and 95% fetal calf serum. Once thawed, the cellsmay be expanded by use of growth factors or stromal cells associatedwith stem cell proliferation and differentiation.

The present invention contemplates also cryopreservation of thechorionic villus and amniotic fluid samples as well as the placentasamples, wherein once thawed, c-kit positive cells can be obtained.

For illustration purposes, c-kit^(pos) cells were induced to differentlineages as described in the Example. The ability to induce specificdifferentiation was initially evident by morphological changes, and wasconfirmed by immunocytochemical and gene expression analyses. Generally,the c-kit positive fetal stem cells can be differentiated into differentcell lineages according to methods well known to one skilled in the art(Stem Cells: Scientific Progress and Future Research Directions.Appendix D. Department of Health and Human Services. June 2001.http://www.nih.gov/news/stemcell/scireport.ht).

Adipogenic specific chemical staining showed that it was possible toinduce lipid accumulation in more than 95% of the c-kit^(pos) chorionicvillus cells when the cells were cultured in specific conditions.Adipocyte induction was confirmed with pparγ2 and LPL expression atdifferent time points.

Consistent with bone differentiation, chorionic and amniotic fetal stemcells showed to be able to produce alkaline phosphatase and to depositcalcium, and the values of both were higher than those reached by adultstem cells under the same conditions. Furthermore, c-kit^(pos) cells inosteogenic media expressed specific genes implicated in mammalian bonedevelopment. Core binding factor A1 (Cbfa1) is an osteoblast specifictranscription factor. Cbfa1 regulates the function of genes expressed inosteoblasts and encodes structural proteins of the bone extracellularmatrix. Forced expression of Cbfa1 in non-osteoblastic cells leads toosteoblast-specific gene expression. Cbfa1 deficient mice and deletionor mutation of the same gene in humans causes cleidocranial dysplasia.

In postnatal life, growth and repair of skeletal muscle are mediated bya resident population of mononuclear myogenic precursors (the “satellitecells”); however their self-renewal potential is limited and decreaseswith age. Previous studies have shown that muscle cells can be derivedfrom mesenchymal stem cells from bone marrow and peripheral tissue. Ithas been shown here that c-kit^(pos) chorionic villus and amniotic cellscan be induced towards muscle differentiation. The c-kit^(pos) cellsformed multinucleated cells that were positive for muscledifferentiation markers (Desmin and Sarcomeric Tropomyosin). Furthermoreby RT-PCR analysis, a characteristic pattern of gene expression,reflecting that seen with embryonic muscle development, wasdemonstrated. Previous studies in mouse embryos have shown that Myf6 isexpressed transiently between days 9 and 11. In our study Myf6 wasexpressed at day 8 and suppressed at day 16. Myf5 in embryonic mousedevelopment is expressed early and continues to be expressed until verylate time points. In our study a low expression of Myf5 was detected inthe induced cells throughout the experiment. Also, as has been shownwith ES cells, MyoD expression was detectable at 8 days in thec-kit^(pos) cells grown under myogenic conditions. Our findingsillustrate that cells derived from chorionic villus, amniotic fluid andplacenta can be induced towards muscle differentiation.

Endothelial cells are usually difficult to isolate and maintain inculture. P1H12, FVIII and KDR are specific markers of endothelialdifferentiation. Amniotic c-kit^(pos) cells cultured in defined mediawere able to form fully differentiated endothelial cells that expressedspecific markers.

In accordance with the present invention, fetal stem cells are obtainedfrom human chorionic villus, amniotic fluid and placenta. Largequantities of chorionic villus, amniotic fluid and placenta cells can beobtained from subjects during pregnancy and/or at birth depending onwhich cell source is used. Fetal stem cells obtained from these sourcesmay be cultured in various media, such as DMEM, F-12, M1 99, RPMI andcombinations thereof, supplemented with fetal bovine serum (FBS), wholehuman serum (WHS), or supplemented with growth factors, cytokines,hormones, vitamins, antibiotics, or any combination thereof. DMEM mediais preferred.

The fetal stem cells may also be expanded in the presence of an agentwhich suppresses cellular differentiation. Such agents are well-known inthe art (Dushnik-Levinson, M. et al., “Embryogenesis in vitro: Study ofDifferentiation of Embryonic Stem Cells,” Biol. Neonate, Vol. 67, 77-83,1995). Examples of agents which suppress cellular differentiationinclude leukemia inhibitory factor (LIF) and stem cell factor. On theother hand, agents such as hydrocortisone, Ca²⁺, keratinocyte growthfactor (KGF), TGF-P, retinoic acid, insulin, prolactin, sodium butyrate,TPA, DIVISO, NMF, DMF, collagen, laminin, heparan SO4, androgen,estrogen, and combinations thereof may be used to induce differentiation(Culture of Epithelial Cells, (R. Ian Freshney ed., Wiley-Liss 1992)).

The cells may be assessed for viability, proliferation potential, andlongevity using standard techniques in the art. For example, atrypanblue exclusion assay, a fluorescein diacetate uptake assay, apropidium iodide uptake assay, or other techniques known in the art maybe used to assess viability. A thymidine uptake assay, an MTT cellproliferation assay, or other techniques known in the art may be used toassess proliferation. Longevity may be determined by the maximum numberof population doublings in extended cultures or other techniques knownin the art.

Additionally, cells of different lineages may be derived by inducingdifferentiation of fetal stem cells and as evidenced by changes incellular antigens. Various differentiation-inducing agents are used toaccomplish such differentiation, such as growth factors (for exampleEGF, aFGF, bFGF, PIDGF, TGF-P), hormones (including but not limited toinsulin, triiodothyronine, hydrocortisone, and dexamethasone), cytokines(for example IL-1α or P, IFN-γ, TFN), matrix elements (for examplecollagen, laminin, heparan sulfate, Matrigel), retinoic acid,transferrin, TPA, and DMSO. Such differentiation-inducing agents areknown to those of ordinary skill in the art (Culture of EpithelialCells, (R. Ian Freshney ed., Wiley-Liss 1992)). Examples below describedifferentiation of fetal stem cells into osteogenic, adipogenic,myogenic and endothelial lineages. Identification of differentiatedcells may be accomplished by staining the cells with tissue-specificantibodies according to techniques known in the art.

In contrast to human embryonic stem (ES) cells whose use has raisedethical concerns, human fetal stem cells of the present invention arederived from a readily available source (chorionic villus or amnioticfluid or placenta) which is normally discarded after birth. Thus,cultured human fetal stem cells are ideal for use in regenerative and/orreconstructive surgery, as well as for use in gene therapy. Somespecific applications of human fetal stem cells are described below.

Fetal stem cells may be used in autologous/heterologous enzymereplacement therapy in specific conditions including, but not limitedto, lysosomal storage diseases, such as Tay-Sachs, Niemann-Pick,Fabry's, Gaucher's, Hunter's, Hurler's syndrome, as well as othergangliosidoses, mucopolysaccharidoses, and glycogenoses.

Additionally, the fetal stem cells of the present invention may be usedas autologous/heterologous transgene carriers in gene therapy to correctinborn errors of metabolism affecting the cardiovascular, respiratory,gastrointestinal, reproductive, and nervous systems, or to treat cancerand other pathological conditions.

Fetal stem cells of the present invention can be used inautologous/heterologous tissue regeneration/replacement therapy,including but not limited to treatment of corneal epithelial defects,cartilage repair, facial dermabrasion, burn and wound dressing fortraumatic injuries of skin, mucosal membranes, tympanic membranes,intestinal linings, and neurological structures. For example,augmentation of myocardial performance can be achieved by thetransplantation of exogenous fetal stem cells into damaged myocardium, aprocedure known as cellular cardiomyoplasty (CCM) which can be used forenhancing myocardial performance and treating end-stage cardiac disease.Fetal stem cells according to the present invention can also be used asa tool for the repair of a number of CNS disorders as described in areview by Cao et al. (Stem cell repair of central nervous system injury,J. Neuroscience Res. 68:501-510, 2002).

Fetal stem cells of the present invention can also be used inreconstructive treatment of damaged tissue by surgical implantation ofcell sheets, disaggregated cells, and cells embedded in carriers forregeneration of tissues for which differentiated cells have beenproduced. The cells may also be used in tissue engineered constructs.Such constructs comprise a biocompatible polymer formed into a scaffoldsuitable for cell growth. The scaffold can be shaped into a heat valve,vessel (tubular), planar construct or any other suitable shape. Suchconstructs are well known in the art (see, e.g., WO02/035992, U.S. Pat.Nos. 6,479,064, 6,461,628).

The amniotic fluid, chorionic villus, placenta tissue and fetal stemcells, before or after differentiation, may be cryopreserved in acryoprotective solution comprising a medium or buffer and acryoprotective agent. Examples of media are Dulbecco's Modified EagleMedium (DMEM), Medium 199 (M199), F-12 Medium, and RPMI Medium. Anexample of a buffer is phosphate buffered saline (PBS). Examples ofcryoprotective agents are dimethylsulfoxide (DMSO) and glycerol.Examples of cryoprotective solutions are: DMEM/glycerol (1:1), DMEM/7.5%DMSO, M199/7.5% DMSO, and PBS/3.5 M DMSO. Optionally, the samples may betreated with antibiotics such as penicillin or streptomycin prior tocryopreservation. Cryopreservation may be accomplished using a rapid,flash-freeze method or by more conventional controlled rate-freezemethods. Rapid freezing of amniotic tissue may be accomplished byplacing sample(s) in a freezing tube containing a cryoprotectivesolution and then rapidly immersing the freezing tube in liquidnitrogen. General slow freezing may be accomplished by placing sample(s)in a freezing tube containing a cryoprotective solution and then placingthe freezing tube in a −70° C. freezer. Alternatively, the sample(s) maybe subjected to controlled rate freezing using a standard cryogenic ratecontrolled system.

Products of fetal stem cells of the present invention may be used inreconstructive treatment, either in vivo or ex vivo. Examples of agentsthat can be produced using fetal stem cells of the present inventioninclude growth factors, cytokines, and other biological responsemodifiers.

The references cited herein and throughout the specification areincorporated by reference in their entirety.

The invention will be further clarified by the following examples, whichare intended to be purely exemplary of the invention.

EXAMPLES

In this example the feasibility of isolating stem cells from humanembryonic and fetal chorionic villi and amniotic fluid was investigated.Discarded cultures of chorionic villi cells and human amniotic fluidcells collected for prenatal diagnostic tests were obtained from morethan 300 human pregnant females ranging from 23 to 42 years of age underan approved institutional Investigation Review Board protocol.

To establish the cultures, human amniotic fluid was obtained bytransabdominal amniocentesis at 14 to 21 weeks of gestation, and humanembryonic chorionic villus tissue specimens were obtained at 10 to 12weeks of gestation through a transabdominal approach.

Amniotic fluid samples were centrifuged and the cell supernatant wasresuspended in culture medium. Approximately 10⁴ cells were seeded on22×22 mm cover slips. Cultures were grown to confluence for about 3 to 4weeks in 5% CO₂ at 37° C. Fresh medium was applied after five days ofculture and every third day thereafter.

Chorionic villus cells were isolated from single villus under lightmicroscopy. The cells were allowed to proliferate in vitro and weremaintained in culture for about 4 weeks. The culture medium consisted ofmodified αMEM (18% Chang Medium B, 2% Chang C with 15% embryonic stemcell-certified fetal bovine serum, antibiotics and L-glutamine) (J. H.Priest, Prenatal Chromosomal Diagnosis and Cell Culture in The ACTCytogenetics Laboratory Manual, Margaret J. Barch (Raven Press, New Yorked. 2, 1991) cap. 5 p. 149).

The cells were sub-cultured using 0.25% trypsin containing 1 mM EDTA for5 minutes at 37° C. Cells were seeded at 3000 cells/cm² in 24 wellplates. Cell numbers were determined after 4, 8, 16, 24 and 32 days inquadruplicate values. For the first time point (4 days), the medium wasremoved from the 24 well plates. The cells were rinsed once withPBS/EDTA, and were incubated with 0.2 ml trypsin/EDTA for 10 minutes at37° C. The cells were resuspended with trypsin/EDTA solution severaltimes to avoid cell clusters, before being transferred to 9.8 ml ofisotonic fluid. Cells were counted as recommended by the manufacturer'sinstructions (Coulter Counter). An MTT assay was performed after 4, 8,16, 24 and 32 days. 100 μl of MTT reagent (Sigma-Aldrich) was added to 1ml of medium for 3 hours. The cells were lysed and color was extractedwith isopropanal containing 0.1M HCl. Extinction was read in a Bioradreader at 570 nm against 655 nm. Results were expressed as a cell count.Growth curves from both cell sources were obtained and the morphology ofthe cells in culture was documented.

Cells from chorionic villi and amniotic fluid underwent phenotypicanalysis. Immunocytochemistry of the amniotic fluid confirmed that mostof the cells were of epithelial origin and stained positively forcytokeratins. Most of the stromal cells stained for α-actin, and only afew cells were positive for desmin or myosin expression (von Koskull,H., et al., Prenat. Diagn., 1(4), p. 259 (1981); Medina-Gomez, P. and T.H. Johnston, Hum. Genet., 60(4), p. 310 (1982)).

The cells were analyzed using FACS for CD34 (Pharmingen International,San Diego, Calif.), CD90 (Santa Cruz Biotechnology, Inc., Santa Cruz,Calif.), CD105 (Pharningen International), CD133 (Miltenyi Biotec,Bergisch Gladbach, Germany), and c-kit (Santa Cruz Biotechnology, Inc).For all antibodies, 0.5×10⁶ of either chorionic villus or amniotic cellswere incubated in 500 μl of primary antibody solution (2% FBS in PBS) ata concentration of 1:100 on ice for 30 minutes. After incubation withthe primary antibodies, the cells were washed twice with 2 ml of 2% FBSin PBS, spun down at 1100 RPM for 7 minutes, and either resuspended in0.5 ml PBS containing 2% FBS, or incubated in the dark, on ice for 30minutes, in 100 μl of FITC labeled secondary antibody (1:100, SouthernBiotechnology Associates Inc., Birmingham, Ala.). The cells were washedtwice with 2 ml of PBS containing 2% FBS, spun down, and resuspended inPBS with 2% FBS for cell analysis. IgG-PE (Pharmingen International) andIgG1κ (unconjugated, Pharmingen International) were used as controls.FACS analysis was performed with a FACScalibur (Becton Dickinson, SanJose, Calif.). Immunocytochemistry was done as follow: cells, grown onchamber slides (Nalge Nunc Int., Naperville, Ill.), were fixed in 4%formaldehyde and in ice-cold methanol. Cell layers were washed with PBS.Cell surface gly-colipid- and glycoprotein-specific mAbs were used at1:15 to 1:50 dilution. MC480 (SSEA-1), MC631 (SSEA-3), and MC813-70(SSEA-4) antibodies were supplied by the Developmental Studies HybridomaBank (University of Iowa, Iowa City). Antibodies were detected usingbiotinylated anti-mouse secondary antibody, strepavi-din-conjugatedhorseradish peroxidase, and 3-amino-9-ethylcarbazole chromagen(BioGenex). Cells prepared for cytogenetic analysis were incubated ingrowth media with 0.1 mg/ml of Colcemid for 3-4 hr, trypsinized,resuspended in 0.075 M KCl, and incubated for 20 min at 37° C., thenfixed in 3:1 methanolyacetic acid.

FACS analyses of the cells showed that between 18% and 21% of the cellsexpressed CD90 and CD105, while much lower percentages of cellsexpressed c-kit, CD34 and AC133 (between 0.8% and 3%). Similar patternsof expression were obtained for the chorionic villus cells.

Cells expressing c-kit (c-kit^(pos)) were successfully immuno-isolatedfrom chorionic villi and were maintained in culture in Chang medium. Thec-kit^(pos) cells expressed human embryonic stage specific markers SSAE3and SSAE4 and did not express mouse embryonic stage specific markerSSAE1 (FIG. 1B-D) (Thomson, J. A., et al., Science, 282(5391), p. 1145(1998)). The c-kit^(pos) cells maintained a round shape when they werecultured in non-treated culture dishes for almost one week and theirproliferative activity was low. After the first week, the cells begun toadhere to the plates and changed their morphology, becoming moreelongated, and proliferating more rapidly. Interestingly andimportantly, no feeder layers were required either for maintenance orexpansion.

In this study c-kit^(pos) cells, obtained from early to late passages,were inducible to different cell lineages including osteogenic,adipogenic, myogenic, neurogenic and endothelial cell lineages underspecific growth factors. The ability to induce specific differentiationwas initially evident by morphological changes, and was also confirmedimmunocytochemically, by gene expression patterns, and by cell-specificfunctional analyses.

Stem cells from bone marrow were purchased (Clonetics) and used as apositive control. The CD34, CD90, CD105 and AC133 immunoisolated cells,and the remaining non-immunoseparated cells did not show anypluripotential capacity. Because amniotic fluid contains both urine andperitoneal fluid, cells isolated from discarded human neonatal urine andperitoneal fluid were used as controls. Human urine and peritonealcontrol fluids did not yield any c-kit^(pos) cells, and the c-kit^(neg)cells did not show any pluripotential ability.

It is known that amniotic fluid, in general, contains very few maternalcells. To determine if any maternal c-kit^(pos) cells were present inthe chorionic villus or amniotic fluid samples, studies were performedusing cells from male fetuses. All the caryotyped c-kit^(pos) cellsshowed an XY karyotype indicating that no C-kit^(pos) maternal cellswere present in the studies samples. C-kit^(pos) cells from femaleembryos and fetuses were used as controls, and they did not show anydifference in their pluripotential ability.

The c-kit^(pos) cells derived from chorionic villi and amniotic fluidshowed a high self-renewal capacity with over 250 population doublings,far exceeding Hayflick's limit. The cells have now been continuouslypassaged for over 18 months and they have maintained theirundifferentiated state. We have also demonstrated that late passagec-kit^(pos) cells maintain their pluripotential capacity and a normalkaryotype after 250 population doublings (FIG. 1E).

Telomerase activity is normally detectable in human germ cells (Thomson,J. A., et al., Science, 282(5391), p. 1145 (1998)), most immortalizedcell lines, and 80-90% of human tumor samples, in which the telomerelength is preserved. We evaluated the telomerase activity in theisolated and cultured c-kit^(pos) cells using the Telomerase RepeatAmplification Protocol (TRAP) assay (FIG. 1F). TRAP analysis (TRAPezekit, Intergenco Pharmaceuticals) was performed as described in themanufacturer's protocol with one modification. CHAP's lysates weresubjected to 36 cycles of PCR amplification after the telomeraseextension step. Low telomerase activity was detected with the TRAP-assayin the amniotic c-kit^(pos) cells (lane 1) compared to the control(lanes 3 and 4). However, after differentiation, the c-kit^(pos) cellsdid not show any telomerase activity (lane 2). To confirm that themeasured telomerase activity was of functional relevance to the isolatedcells, the telomere length of the c-kit^(pos) cells at early and latepassages were determined by terminal restriction fragment (TRF)analyses. Total cellular DNA was isolated by the DNeasy Tissue Kit(Qiagen Corp) and 2 μg was used for Southern Blot analysis of TRFlengths (TeloTAGGG Telomere Length Assay, Roche Molecular) as describedin the manufacturer's protocol. Briefly, purified genomic DNA wasdigested with a mixture of frequently cutting restriction enzymes. Theresulting fragments were agarose gel electrophoresed and transferred toa nylon membrane by Southern blotting. Hybridization to a digoxigenin(DIG)-labeled probe specific for the telomeric repeats was followed bychemiluminescent detection and exposure of the membrane toautoradiography film. TRF qualitative analysis demonstrated that thec-kit^(pos) cells had similar telomere lengths, both at early (mean TRFlength approximately 20 kb) and late (mean TRF length approximately 20kb) passages (FIG. 1G).

However C-kit^(pos) cells derived from chorionic villi and amnioticfluid expanded clonally more than 250 population doublings whilemaintaining approximately the same telomere length and had additionallyacquired telomerase activity.

This phenomenon suggests that the cell population could have analternative mechanism for lengthening telomeres (ALT) (16, 17). Onepossible answer could be derived from the clonal fluctuations. Sometested clones could have been overlengthned by the action oftelomere-lengthening by an unknown mechanism. Regulation factors couldtherefore influence the activation and inactivation of the telomerasewithout influencing the telomere length (Brayan, T. M et al (1998)Telomere length dynamics in telomerase-positive immortal human cellpopulation). The explanation of this particular phenomenon is not clearand the mechanism for the longevity of these cells in culture isunknown.

To prove the capacity of the c-kit^(pos) cells isolated from amnioticfluid and chorionic villi to differentiate into various cell lineages weused a method of retroviral marling. CKit^(pos) cells were transducedwith a puc-CMMP-IC-eGFP retrovirus and expanded. The infected-cKit^(pos)were sorted by FACS-Excalibur and single eGFP⁺-cells were plated perwell in a 96 wells-plate and expanded. The derived clones were sortedone more time by FACScan instrument (Becton Dickinson, San Jose, Calif.)in-line with a Power Macintosh computer using the CELLQuest software inorder to obtain a subpopulation of clones. The DNA from the originalclones and derived subclones was extracted using a Dnaesy Tissue Kit(Qiagen) and the concentration was measured with a Spectrofotometer(Spectronic 601). Three samples of genomic DNA for each clone andsubclone were digest for three hours with a different mix of restrictionenzymes (mix1. SapI, MfeI, HpaI, DraIII; mix2. BamHI, NheI, HindIII,XhoI, PacI; mix3. BglII, AseI). The fragments were separated byelectrophoresis and transfer by capillarity to a naylon membrane. AneGFP-cDNA probe was constructed from a plasmid (PEGFP-N1 . . . )digesting the plasmid with AgeI and NotI. The fragment was separated byelectrophoresis and the digested DNA was extract with a Gel ExtractionKit (Qiagen) and labelled with digoxigenin for detection with alkalinephosphatase metabolising CDP-Star, a highly sensitive chemiluminescencesubstrate (DIG High Prime DNA Labeling and Detection Starter Kit II,Roche). The blotted DNA fragments were hybridised to the Dig-labelledeGFP cDNA probe and the retrovirus insertion was determined by detectionexposing the membrane to X-ray film.

All experiments were performed with c-kit^(pos) cells obtained fromtwelve clonal cell populations, according to their gestational age (10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21 weeks). Furthermore, allexperiments were also performed with five single c-kit^(pos) clonal cellpopulations obtained from a single fetus (11, 14, 16, 18 and 20 weeks ofgestation). Cells from the different clones showed a similar morphologyand growth behavior. Cells from all clones underwent osteogenic,adipogenic, myogenic, neurogenic and endothelial differentiation. Nostatistical differences were noted in the ability of the 17 clonal cellpopulations to differentiate into separate lineages.

Osteogenic induction. For the induction of osteogenic differentiation,c-kit^(pos) cells isolated from amniotic fluid and chorionic villi werecultured in a defined osteogenic medium. For the induction of osteogenicdifferentiation, the cells were seeded at a density of 3000 cells/cm²and were cultured in DMEM low glucose medium (Gibco/Brl) with 10% fetalbovine serum (FBS, Gibco/Brl), 1% antibiotics (Gibco/Brl), andosteogenic supplements [100 nM dexamethasone (Sigma-Aldrich), 10 mMbeta-glycerophosphate (Sigma-Aldrich), and 0.05 mM ascorbicacid-2-phosphate (Wako Chemicals, Irving, Tex.)]. Jaiswal, N., et al.,J. Cell. Biochem., 64(2), p. 295 (1997).

Control medium was essentially modified αMEM. Medium was changed aboutevery 3 days. Light microscopy analysis of the cells showed that, within4 days in the osteogenic medium, c-kit^(pos) cells lost their spindleshape phenotype (FIG. 2A) and developed an osteoblastic-like appearancewith fingerlike excavations into the cytoplasm (FIG. 2B). At day 16, thecells aggregated, showing typical lamellar bone-like structures.Consistent with bone differentiation, the c-kit^(pos) cells culturedunder osteogenesis inducing conditions produced alkaline phosphatase(AP) and showed calcium deposits. Interestingly, both the amount of APproduction and calcium deposition was higher than those reached by adultosteogenic stem cells cultured under the same conditions. AP activitywas measured using a quantitative assay for p-Nitrophenyl, which isequivalent to AP production. Alkaline phosphatase enzyme cell activitywas measured in quadruplicate cultures. After rinsing with PBS, thecells were incubated with 2-amino-2-methyl-1-propanol buffer, pH 10.3(Sigma-Aldrich #221/26) with 40 mg p-nitrophenyl phosphate(Sigma-Aldrich #104/40) added, at 37° C. for 3 to 35 min. AP activitywas calculated after measuring the absorbance of p-nitrophenyl productsformed at 405 nm on a micro plate reader (Molecular Devices, Spectra MaxPlus). As a standard, p-nitrophenyl standard solution (Sigma-Aldrich#104-1) diluted in 2-amino-2-methyl-1-propanol buffer in concentrationsfrom 0 to 100 nMol p-nitrophenyl was used. Enzyme activity was expressedas nMol p-Nitrophenyl/min/10⁶ cells.

Histochemical Analyses. Alkaline phosphatase activity was determinedhistologically in cells according to the manufacturer's instructions(Sigma-Aldrich Kit #85). Briefly, cells were fixed in a citrate-acetonesolution. An alkaline-dye mixture (fast blue RR solution with naphtholAS-MX phosphate alkaline solution) was added to the cells in the 35 mmculture dishes. The cell cultures were protected from direct light.Prior to viewing, the cell cultures were rinsed with deionized water andair-dried. AP production in the c-kit^(pos) cells grown inosteogenic-inducing medium increased by a factor of 250 compared toc-kit^(pos) cells grown in control medium and c-kit^(neg) cells grown inosteogenic medium at days 16 and 24 (FIG. 2C).

A major feature of osteogenic differentiation is the ability of thecells to precipitate calcium. Cell associated mineralization may beanalyzed using von Kossa staining and by measuring calcium content inthe cells in culture. Von Kossa staining of cells grown in theosteogenic medium showed enhanced silver nitrate precipitation by day16, indicating high levels of calcium. The presence of mineralization incell culture was determined by von Kossa staining. The cell cultureplates were fixed with 10% formaldehyde for 1 h, incubated with 2%silver nitrate solution for 10 min in the dark, washed thoroughly withdeionized water, and then exposed to UV-light for 15 min. Calciumcontent continued to increase exponentially at 24 and 32 days. Incontrast, cells in the control medium did not show any silver nitrateprecipitation (FIG. 2G).

Calcium deposition by the cells was also measured with a quantitativechemical assay which measures calcium-cresolophthalein complexes. Cellsundergoing osteogenic induction showed a significant increase in calciumprecipitation after 16 days (up to 4 mg/dl). The precipitation ofcalcium increased up to 70 mg/dl at 32 days. In contrast, cells grown inthe control medium did not show any increase in calcium precipitation(1.6 mg/dl) by day 32 (FIGS. 2H and I).

C-kit^(pos) cells in osteogenic medium expressed specific genesimplicated in mammalian bone development (AP, core binding factor A1(cbfa1), and osteocalcin) (FIG. 2C). RNA was isolated from culturedcells and cell pellets with RNAzol reagent (Tel-Test Inc., Friendswood,Tex.) according to the manufacturer's protocol. RNA (2 μg) was processedfor c-DNA synthesis with Superscript II reverse transcriptase withrandom hexamers (Life Technologies). C-DNA was used for each PCRreaction, in a final volume of 30 μl with 200 nM dNTP, 10 pM of eachprimer, 0.3 U Taq-DNA-polymerase, reaction buffer, and MgCl2 (LifeTechnologies), in a PTC-100 cycler (MJ-Research Inc., Watertown, Mass.).The cycling conditions consisted of 94° C. for 2 minutes, annealing at63° C. for 40 seconds, and elongation at 72° C. for 1 minute. Cyclenumbers varied between 22 and 37 cycles and were chosen in theexponential phase of the RT-PCR reaction. Primer sequences and fragmentsizes are listed in Table 1. All primers were obtained from LifeTechnologies. Primers for human core binding factor A1 (cbfa1) primers(sense 5′GGCCTTCCACTCTCAGTAAGA3′ (SEQ ID NO:1) and antisense5′GATTCATCCATTCTGCCACTA3′, (SEQ ID NO:2)28 cycles at 63° C.) amplified afragment of 474 bp and human osteocalcin (sense5′CCCTCACACTCCTCGCCCTAT3′ (SEQ ID NO:3) and antisense5′GGTAGCGCCTGGGTCTCTTCA3′, SEQ ID NO:4) amplified a fragment of 144 bp.Human peroxisome proliferator-activated receptor γ2 (pparγ2) primers(sense 5′TGAACGACCAAGTAACTCTCC3′ (SEQ ID NO:5) and antisense 5′CTCATGTCTGTCTCCGTCTTC3′, (SEQ ID NO:6) 29 cycles at 64° C.) yielded afragment of 460 bp. 533 bp. Human lipoprotein lipase (lpl) primers(sense 5′CTGGTCGAAGCATTGGAAT3′ (SEQ ID NO:7) and antisense5′TGTAGGGCATCTGAGAACGAG3′, (SEQ ID NO:8) 29 cycles at 64° C.) amplifieda fragment of 366 bp. Human Myogenic Regulatory Factor 4 (MRF4) (sense5′CGACAGCAGCGGAGAGG3′ (SEQ ID NO:9) and antisense5′GGAATGATCGGAAACACTTGG3′, (SEQ ID NO:10) 37 cycles at 62° C.) wasdetected as band of 421 bp and human myoD (sense TCCGCGACGTAGACCTGAC3′(SEQ ID NO:11) and antisense 5′GATATAGCGGATGGCGTTGC3′, SEQ ID NO:12)amplified a segment of 449 bp. Human desmin primers (sense5′CCATCGCGGCTAAGAACATT3′(SEQ ID NO:13) and antisense5′TCGGAAGTTGAGGGCAGAGTA3′, (SEQ ID NO:14) 27 cycles at 62° C.) amplifieda fragment of 440 bp, Primers for human β2-microglobulin (β2-MG) (sense5′GCTCGCGCTACTCTCTC3′ (SEQ ID NO:15) and antisense5′TTAACTATCTTGGGCTGTGAC3′, (SEQ ID NO:16) 23-26 cycles at 62-64° C.)amplified a fragment of 315 bp. Primers for human CD106 (VCAM) (sense5′TCCAGCGAGGGTCTACCAG3′ (SEQ ID NO:16) antisense5′TGTTTGCGTACTCTGCCTTTG3′, SEQ ID NO:17) amplified a segment of 774 bpand human CD31 (PECAM) (sense 5′CCTTCTCTACACCCAAGTTCC3′ (SEQ ID NO:18)and antisense 5′GAAATAGGCAAAGTTCCACTG3′, SEQ ID NO:19) yielded afragment of 628 bp.

C-kit^(pos) cells grown in osteogenic medium showed an activation of theAP gene at each time point. No transcription of the AP gene was detectedat 8, 16, 24 and 32 days in the c-kit^(pos) cells grown in controlmedium. Expression of cbfa1, a transcription factor specificallyexpressed in osteoblasts and hypertrophic chondrocytes that regulatesgene expression of structural proteins of the bone extracellular matrixin osteoblasts (24, 25), was highest in cells grown in osteogenicinducing medium at day 8 and decreased slightly at days 16, 24 and 32.The expression of cbfa1 in the controls was significantly lower at eachtime point. Osteocalcin was expressed only in the c-kit^(pos) cells inosteogenic conditions at 8 days. No expression of osteocalcin wasdetected in the c-kit^(pos) cells in the control medium and c-kit^(neg)cells in osteogenic medium at any time point.

C-kit^(pos) cells were also seeded on hydroxyapatite-collagen scaffolds(Collagraft, Neucoll, Zimmer, Warsaw, Indiana) at a density of 10×10⁶cells/cm². Cells were induced into an osteogenic lineage in a bioreactorfor 16 d. The rods were implanted subcutaneously in athymic mice,harvested after 4 and 8 weeks, and analyzed. Bone-like tissue wasevident, surrounded by an extracellular matrix which stained blue withMasson's trichrome. Toluidine blue staining confirmed the osteogenicphenotype. Small calcified areas within the implanted tissue stainedpositively with von Kossa, indicating bone formation. Unseededconstructs, as controls, showed only a few infiltrating cells, and nobone-like structures were noted (FIG. 2).

Adipogenic induction. To promote adipogenic differentiation, we culturedthe c-kit^(pos) cells in defined adipogenic medium. For the induction ofadipogenic differentiation, the cells were seeded at a density of 3000cells/cm² and were cultured in DMEM low glucose medium with 10% FBS, 1%antibiotics, and adipogenic supplements [1 μM dexamethasone, 1 mM3-isobutyl-1-methylxanthine (Sigma-Aldrich), 10 μg/ml insulin(Sigma-Aldrich), and 60 μM indomethacin (Sigma-Aldrich)].

Control medium consisted of modified αMEM. Medium changes were performedevery 3 days. C-kit^(pos) cells cultured with adipogenic supplementschanged their morphology from elongated to round within 8 days. Thiscoincided with the accumulation of intracellular triglyceride droplets[FIG. 3A]. The presence of adipose elements in cell culture wasdetermined with Oil-O-Red staining. The 2 well chamber slides werewashed in deionized water and air-dried. The cells were incubated withoil red O staining solution for 15 minutes, rinsed with 50% ethanol 3times, rinsed with distilled water, counterstained with Gillshematoxilin for 30 sec to 1 min, and rinsed in deionized water 3 to 4times. After 16 days in culture, more than 95% of the cells had theircytoplasm filled with lipid-rich vacuoles, which stained with Oil-O-Red(FIG. 3B).

Chamber slides were mounted with water-based mounting media. Thec-kit^(pos) cells cultured in control medium and the c-kit^(neg) cellscultured in adipogenic medium did not show any phenotypic changesconsistent with adipogenic differentiation and did not stain withOil-O-Red (FIG. 3C).

Adipogenic differentiation was confirmed by RT-PCR analysis. We analyzedthe expression of peroxisome proliferation-activated receptor γ2(pparγ2) (28, 29) a transcription factor that regulates adipogenesis,and of lipoprotein lipase. Expression of these genes was upregulated inthe c-kit^(pos) cells under adipogenic conditions. C-kit^(pos) cellscultured under control conditions and c-kit^(neg) cells cultured underadipogenic conditions did not express either gene at any time point[FIG. 3D].

C-kit^(pos) cells were seeded on polyglycolic acid (PGA) polymerscaffolds at a density of 10×10⁶ cells/cm². Cells were induced into anadipogenic lineage in a bioreactor for 16d. The scaffolds were implantedsubcutaneously in athymic mice, harvested after 4 and 8 weeks, andanalyzed. The retrieved scaffolds showed the formation of fatty tissuesgrossly. The presence of adipose tissue was confirmed with Oil-O-Redstaining [FIG. 3].

Myogenic induction. In postnatal life, growth and repair of skeletalmuscle are mediated by a resident population of mononuclear myogenicprecursors (satellite cells); however their self-renewal potential islimited and decreases with age. Previous studies have shown that musclecells can be derived from mesenchymal stem cells from bone marrow andperipheral tissue (30). In this study, c-kit^(pos) cells were inducedtowards muscle differentiation. We seeded c-kit^(pos) cells in 35 mmplates precoated with Matrigel in a defined medium. The defined myoblastgrowth medium consisted of DMEM low glucose containing 10% horse serum(GIBCO, BRL), 0.5% chick embryo extract (GIBCO, BRL) and 1%penicillin/streptomycin (GIBCO, BRL) (Reddel, R. R. et al., (1997).Immortalized cells with no detectable telomerase activity. Biochemistry62, 1254-1262). Matrigel (Collaborative Biomedical Products, UniversalBiologicals Ltd.) was diluted in DMEM to 1 mg/ml, plated and incubatedfor 1 h at 37° C., before the cells were seeded. Rosenblatt, J. D., etal., In Vitro Cell Dev. Biol. Anim., 31(10), p. 773 (1995). Definedmedium containing 5-azacytidine was added after 12 hours and replaced 24hours later with 5-azacytidine-free defined medium. As a control,undifferentiated cells were grown in 35 mm plates with modified αMEM.Medium changes were performed every 3 days.

Induction with 5-azacytidine for 24 hours induced the formation ofmultinucleated cells after a 24 to 48 hour period [FIG. 4A]. Themultinucleated cells expressed the muscle differentiation markers desminand sarcomeric tropomyosin. [FIGS. 4D and F]. C-kit^(pos) cells grown incontrol medium and c-kit^(pos) cells grown in myogenic conditions didnot lead to cell fusion or multinucleated cells.

We analyzed the expression of MyoD, Myf 6 (MRF4) and Desmin in cellsundergoing myogenic differentiation, using RT-PCR. A characteristicpattern of gene expression, reflecting that seen with embryonic muscledevelopment, was demonstrated [FIG. 4G] (32, 33). Previous studies inmouse embryos have shown that Myf6 is expressed transiently between days9 and 11 (34, 35). In our study Myf6 was expressed at day 8 andsuppressed at day 16. As has been shown with ES cells, MyoD expressionwas detectable at 8 days and suppressed at 16 days in the c-kit^(pos)cells grown under myogenic conditions. Desmin expression was induced at8 days and increased by 16 days in the c-kit^(pos) cells cultured inmyogenic medium. In contrast, there was no activation of Myf6, MyoD orDesmin in the control cells at 8 and 16 days.

C-kit^(pos) cells were labeled with a fluorescence marker (PKH26 GreenFluorescent Cell Linker, Sigma-Aldrich) and were induced into a myogeniclineage. The myogenic cells were resuspended in rat tail collagencontaining 17% Matrigel (BD Biosciences), were injected into thehindlimb musculature of athymic mice, and were retrieved after 4 weeks.The injected myogenic cells showed the formation of muscle tissue whichexpress desmin (A) and maintained its fluorescence (B) [FIG. 4].

Endothelial induction. To induce endothelial differentiation, we platedthe cells in dishes precoated with PBS-gelatin. The cells weremaintained in culture for 1 month in endothelial-defined medium. Toinduce endothelial differentiation, the cells were plated at a densityof 3000 cells/cm² in 35 mm dishes precoated with PBS-gelatin. The cellswere maintained in culture for 1 month in endothelial basal medium-2(EBM-2 Clonetics BioWittaker inc., Walkersville, Md.) supplemented with10% FBS (GIBCO/BRL, Grand Island, N.Y.), 1% antibiotics (GIBCO/BRL,Grand Island, N.Y.) and 1% L-glutamine (GIBCO/BRL, Grand Island, N.Y.).Basic fibroblast growth factor (bFGF) was added every other day. After 1week in culture the c-kit^(pos) cells changed their morphology, and bythe second week, the cells were mostly tubular [FIG. 5A]. Human-specificendothelial cell For hepatic differentiation, c-kit^(pos) cells surfacemarkers (P1H12), factor VIII (FVIII) and KDR are specific fordifferentiated endothelial cells. The differentiated cells stainedpositively for FVIII, KDR and P1H12 [FIG. 5B-D]. C-kit^(pos) cellscultured in Chang medium for the same period of time were not able toform tubular structures and did not stain for endothelial specificmarkers. Endothelial cells are usually difficult to isolate and maintainin culture. In our study the endothelial cells, once differentiated,were able to grow in culture and formed capillary-like structures invitro [FIG. 5E]. In order to confirm the phenotypic changes we performedRT-PCR. Platelet endothelial cell adhesion molecule 1 (PECAM-1 or CD31)and vascular cell adhesion molecule (VCAM) were markedly increased inthe ckit^(pos) cells induced in endothelial media but were not amplifiedin the ckit^(pos) cells cultured in control media [FIG. 5F].

Hepatic induction. For hepatic differentiation, c-kit^(pos) cells fromamniotic fluid and chorionic villi, seeded in Matrigel coated dishes,were cultured in hepatic condition for 9 days. The c-kit^(pos) cellsseeded in Matrigel (Collaborative Biomedical Products, UniversalBiologicals Ltd.) using a modified manufacturer thin gel method using100 ul/cm² surface. The cells seeded in 24-well plates at a density of25,000 cells/cm² were allowed to establish themselves in this culture inChang medium for 3 days to achieve a semi-confluent density.Differentiation was induced in three steps. The base medium consisted oflow glucose Dulbecco's medium (Gibco/Brl) containing 300 uMmonothioglycerol (Sigma-Aldrich), 100 U/ml penicillin, and 100 U/mlstreptomycin (Gibco/Brl) with 15% fetal bovine serum FBS, (Gibco/Brl).Cells were grown initially for 3 days in the presence of 100 ng/mlacidic fibroblastic growth factor ( ). This step was followed byexposure to 20 ng/ml hepatocyte growth factor ( ) for 3 days andconcluded with 20 ng/ml hepatocyte growth factor, 10 ng/ml oncostatin M( ), 10-7 M dexamethasone (Sigma-Aldrich)¹. Cells were maintained in thesame media used for end stage differentiation. Control cell populationswere seeded in the same manner as differentiated cells, but were simplymaintained in control medium. After the differentiation process thecells were maintained in culture for 30 days.

In order to evaluate the hepatic differentiation, the expression ofalbumin was evaluated and the urea production was measured using astandard urea nitrogen essay in the differentiated cells and in thecontrol cells. Cells suspended in matrigel were trypsanized for 10minutes with light mechanical assistance and cytospin onto slides at adensity of 1000 cells/slide. Cells were probed for albumin with goatanti-human albumin ( ) using standard immunocytochemistry protocol withDAPI nuclear counterstain. Urea production was measured using acolorometric urea nitrogen assay (Sigma-Aldrich). Differentiated orcontrol cell populations were placed in ammonium chloride at asupraphysiological level of 20 mM NH4Cl to examine maximum rate of ureaproduction of each of these cell types. The medium was then collectedafter 30 minutes of exposure and tested per manufacturer instructionswith and without urease to obtain true levels of urea. After 7 days ofthe differentiation process, cells tended to show morphological changesfrom elongated fibroblastic cells to more epitheliod cobblestoneappearances. Cells showed positive staining for at day 12 postdifferentiation. The maximum rate of urea production for hepaticdifferentiation induced cells was 4.7×10⁻⁴ μg urea/hour/cell as opposedto 2.36×10⁻⁴ μg urea/hour/cell for control cell populations.

Neurogenic induction. For neurogenic induction, we cultured amniotic andchorionic villi ckit^(pos) cells in defined neurogenic medium (40, 41).For neurogenic induction, the amniotic cells were seeded at aconcentration of 3000 cells/cm² in 100 mm plates and were cultured inDMEM low glucose medium (GIBCO/BRL, Grand Island, N.Y.), 1% antibiotics(GIBCO/BRL, Grand Island, N.Y.), 2% DMSO and 200 μM butylatedhydroxyanisole (BHA, Sigma-Aldrich, St. Louis, Mo.). Neuron growthfactor (NGF) (8 μl/ml) was added to the culture every 2 days. After 2days the medium was changed to control medium and the same amount of NGFwas continuously supplemented. Cells were fixed for immunocytochemestryat 4 and 8 days.

After 2 days the medium was changed to control medium and the sameamount of NGF was continuously supplemented. Cells were fixed forimmunocytochemestry at 4 and 8 days. Control medium consisted ofmodified αMEM. Medium changes were performed every 3 days.

C-kit^(pos) cells cultured in neurogenic conditions changed theirmorphology within the first 24 hours. Two different cell populationswere apparent, morphologically large flat cells and small bipolar cells.The bipolar cell cytoplasm retracted towards the nucleus, formingcontracted multipolar structures. Over the subsequent hours, the cellsdisplayed primary and secondary branches and cone-like terminalexpansions [FIG. 6A]. Induced C-kit^(pos) cells showed a characteristicsequence of expression of neural-specific proteins. At an early stagethe intermediate filament protein nestin (BD Bioscience), which isspecifically expressed in neuroepithelial stem cells, was highlyexpressed [FIG. 6B]. The expressions of β III tubulin [FIG. 6C] andglial fibrillary acidic protein (GFAP) (Santa Cruz) [FIG. 6D], markersof neuron and glial differentiation (42), respectively, increased overtime and seemed to reach a plateau at about 6 days. C-kit^(pos) cellscultured in Chang medium and c-kit^(neg) cells cultured in neurogenicmedium for the same period did not stain for neurogenic specificmarkers. Furthermore we analyzed the functional behavior of the neuronalcells. The C-kit^(pos) cells cultured under neurogenic conditions showedthe presence of the neurotransmitter glutamic acid in the collectedmedium. Glutamic acid is usually secreted by fully differentiatedneurons in culture (43). Non-induced cells, heat inactivated cells andcontrol urothelial cells did not secrete any glutamic acid [FIG. 6E].

Hematopoietic differentiation. For the hematopoietic differentiation weused a liquid media (StemSpan by STEM CELL TECHNOLOGIES, seewww.stemcell.com). The following frowth factors were added to the cellculture medium: stem cell factor, GM-CSF, IL6, IL3, G-CSF according tothe manufacturer's instructions (STEM CELL TECHNOLOGIES). Thehematopoietic differentiation was assessed by analyzing the cellmorphology.

Murine chorionic villi and amniotic fluids were collected from femaleC57BL/6 mice that were from 6 to 9 weeks of age and that were 12 dayspregnant (protocol approved Animal Care and Use Committee, Children'sHospital, Boston) under light microscopy. The samples were proceeded aspreviously described. Briefly placentas were dissected under lightmicroscope and the chorionic villi were explanted. Chorionic villi andamniotic fluid derived cells were cultured in the same conditions usedfor human cells with addition of LIF (10 ng/ml) (Sigma-Aldrich). Theckit^(pos) cells were transduced with a puc-CMMP-IC-eGFP retrovirus andexpanded. The infected-cKit^(pos) were sorted by FACS-Excalibur and asingle eGFP⁺-cells was plated per well in a 96 wells-plate and expanded.

In order to assess the ability of these cells to contribute to differenttissue 10-12 ckit^(pos) infected cells were microinjected into 4-day-oldblastocysts of C57BL/6-TgN(lacZpl)60Vij. The blastocysts weretransferred to foster mothers and mice were allowed to develop until 16days of gestation.

The fetuses were collected, embedded in OCT and 10 μm whole-bodysections were prepared. Tissue sections were stained for β-galactosidaseenzyme activity and observed under fluorescent microscope in order toidentify the c-kit^(pos) cells carrying the gene for the green protein.

We also collected multiple organs, they were embedded in OCT and 5 μmsections were prepared as described. The sections were stained forβ-galactosidase enzyme activity and observed.

Frozen section were cut at 10 μm and fixed with 2% formaldehyde, 0.2%glutaraldehyde, 0.02% NP-40 and 0.01% sodium deoxycolate in PBS pH7.8for 30 min at RT and then wash 3 times with PBS. Samples were incubatedin LacZ staining solution (2 mM MgCl2, 0.02% NP-40, 0.01% sodiumdeoxycolate, 5 mM K-ferricyanide, 5 mM K-ferrocyanide and 0.1% X-gal inPBS pH7.8) at 37 C for 8 to 16 hours in dark. Images were acquired usingIX-70 microscope with Magna Fire Digital Imaging Camera System (Olympus)and processed using Adobe Photoshop 5.0.

Discussion. Stem cells have been reported to exist during embryonicdevelopment and postnatally, in bone marrow, skeletal muscle and skin(for a recent review discussion of stem cells see, J. Pathol, Vol 197,Issue 4, 2002). Embryonic stem (ES) cells are derived from the innercell mass (ICM) at the blastula stage. ES cells tend to differentiatespontaneously into various types of tissues. However, isolation of thesecells, particularly from human embryos, has resulted in heated debateabout ethical concerns of this procedure which results in destruction ofthe embryo.

Adult stem cells do not differentiate spontaneously, but can be inducedto differentiate by applying appropriate growth conditions. Adult stemcells seem to be easier to maintain in culture than ES cells. However,adult stem cells have the disadvantage of not being immortal, and mostof them lose their pluripotency after a defined number of passages inculture. This short life-span is a significant obstacle in clinicalapplications where a large amount of cells are needed.

Fetal tissue has been used in the past for transplantation and tissueengineering research because of its pluripotency and proliferativeability. Fetal cells have a higher proliferative capacity than adultcells and may preserve their pluripotency longer in culture. However,there are several issues concerning the availability of fetal celltransplants. Beyond the ethical concerns regarding the use of cells fromaborted fetuses or living fetuses, there are other issues, which remaina challenge. For example, previous studies have shown that up to sixfetuses are required to provide enough material to treat one patientwith Parkinson's disease (45).

This invention is based upon a finding that chorionic villi and amnioticfluid cells, which have been used for decades for prenatal diagnosis,represent a viable source of human fetal stem cells from both embryonicand fetal sources and can be used therapeutically. It is well known thatboth chorionic villi tissue specimens and amniotic fluid contain a largevariety of cells. The vast majority of the cells collected fromchorionic villi and amniotic fluid are already differentiated, andtherefore have a limited proliferative ability (46). We have hereidentified and isolated cells that maintained both their pluripotentialand proliferative ability.

Many efforts in the past were aimed at trying to identify antibodiesthat bind cell surface markers on undifferentiated cells. C-kit, CD105,CD34 and CD90 have been identified as potential stem cell markers. Wefound that less than 1% of the embryonic and fetal cells isolated fromchorionic villi and amniotic fluid were c-kit^(pos) and that only theisolated c-kit^(pos) cells had the pluripotent phenotype. The c-kit geneencodes for a tyrosine kinase growth factor receptor for Stem CellFactor (SCF), also called mast cell growth factor that is essential forhematopoesis, melanogenesis and fertility (46). The Kit protein (CD117)is constitutively expressed in hematopoetic stem cells, mast cells, germcells, melanocytes, certain basal epithelial cells, luminal epitheliumof breast, and the interstitial cells of Cajal of the gastrointestinaltract (47). The c-kit gene plays a fundamental role during theestablishment, maintenance and function of germ cells (48). In theembryonal gonad, the c-kit receptor and its ligand SCF are required forthe survival and proliferation of primordial germ cells. Furthermorerecent studies have shown that c-kit is expressed in placental tissueduring pregnancy. C-kit and SCF may have an important role in embryonicdevelopment as evidenced by expression and localization at thefeto-maternal interface (49). In the postnatal animal, c-kit/SCF arerequired for production of the mature gametes in response togonadotropic hormones, i.e. for the survival and/or proliferation of theonly proliferating germ cells of the testis, the spermatogonia, and forthe growth and maturation of the oocytes. Experiments in vitro haveshown that c-kit is a potent mitogen for primitive hematopoetic cells.In mice, loss of either SCF or c-kit results in macrocytic anemia,leading to death in-utero or within the first postnatal days.

Adult stem cells have a limited capacity to proliferate and they undergosenescence when the Hayflick limit is reached. Furthermore, the adultstem cells are not able to preserve their ability to differentiate intomultiple lineages after a few passages. Contrary to adult stem cells,embryonic stem (ES) cells have an unlimited capacity to proliferate andthey are able to maintain their potential for differentiation inculture. We found that the c-kit^(pos) cells derived from humanembryonic and fetal chorionic villi and amniotic fluid were pluripotentand were able to differentiate into osteogenic, adipogenic, myogenic,neurogenic, hepatic and endothelial phenotypes. The possibility offorming different types of tissues was confirmed in vivo. The cells weretelomerase positive, highly clonogenic, and the cloned fetal stem celllines were able to undergo more than 250 cell divisions, exceedingHayflick's limit. The stem cell lines maintained their telomere lengthand differentiation potential in culture, even after 250 populationdoublings. In addition, the c-kit^(pos) cells did not require a feederlayer for growth. The c-kit positive human fetal stem cells alsoexpressed markers known to be associated with human embryonic stem cells(SSAE3 and SSAE4).

In conclusion, we describe the isolation, expansion and differentiationof stem cells from human embryonic and fetal chorionic villi andamniotic fluid. These cells provide an excellent source for bothresearch and therapeutic applications. Embryonic and fetal stem cellshave a better potential for expansion than adult stem cells and for thisreason they represent a significantly better source for therapeuticapplications where large numbers of cells are needed.

Further, the ability to isolate stem cells during gestation may also beadvantageous for treatment of fetuses with congenital malformations inutero. When compared with ES cells, c-kit^(pos) fetal stem cellsisolated from chorionic villi and amniotic fluid have many similarities:they can differentiate into all three germ layers, they express commonmarkers and show telomerase activity. However c-kit^(pos) cells isolatedfrom the chorionic villi and amniotic fluid have considerable advantagesover ES cells. The c-kit^(pos) cells isolated from the chorionic villiand amniotic fluid easily differentiate into specific cell lineages,they do not need feeder layers to grow, and most importantly, theisolation of these cells does not require the sacrifice of human embryosfor their isolation, thus avoiding the current controversies associatedwith the use of human embryonic stem cells.

The references cited herein and throughout the specification areincorporated by reference in their entirety.

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1. A method of proliferating a population of cells enriched for humanpluripotent fetal stem cells comprising: (a) selecting at least onec-kit positive cell from a human amniotic fluid sample, wherein saidcell is c-kit, SSEA3 and SSEA4 positive, and immunonegative for SSEA1;(b) introducing said at least one selected cell to a culture medium; and(c) proliferating said at least one selected cell in the culture medium.2. A method of obtaining a composition enriched for human pluripotentfetal stem cells, said cells being c-kit, SSEA3 and SSEA4 positive, andSSEA1 negative, comprising the steps of: (a) cryopreseverving a specimenof the amniotic fluid; (b) thawing the cryopreserved specimen at a laterdate; and (c) selecting for c-kit positive cells.
 3. A method ofproducing a composition enriched for human pluripotent fetal stem cells,said cells being c-kit, SSEA3 and SSEA4 positive, and SSEA1 negativecomprising, (a) isolating c-kit positive cells from a sample of amnioticfluid; and (b) proliferating said cells in culture medium.
 4. A methodof obtaining a composition enriched for human pluripotent fetal stemcells that are c-kit, SSEA3 and SSEA4 positive, and SSEA1 negativecomprising selecting c-kit positive cells from a human amniotic fluidsample.
 5. The method of claim 4, wherein the selecting c-kit positivecells is performed using an antibody against c-kit.
 6. The method ofclaim 5, wherein the antibody against c-kit is a monoclonal antibody. 7.The method of claim 5, wherein the monoclonal antibody against c-kit isa mouse monoclonal IgG against an antigenic epitope of human c-kit. 8.The method of claim 5, wherein the antibody against c-kit isfluorochrome conjugated.
 9. The method of claim 5, wherein the antibodyagainst c-kit is conjugated to magnetic particles.
 10. The method ofclaim 4, wherein the selecting is by flow cytometry.
 11. The method ofclaim 4, wherein the selecting is by fluorescence activated cell sortingor high gradient magnetic selection.
 12. The method of claim 4, whereinthe amniotic fluid sample is cryopreserved prior to the selection step.13. The method of claim 4, further comprising cyropreserving the c-kit,SSEA3 and SSEA4 positive, and SSEA1 negative cells.
 14. A method ofproducing a composition enriched for human pluripotent fetal cellscomprising: (a) selecting at least one c-kit positive cell from a humanamniotic fluid sample, wherein said cell is c-kit, SSEA4, and telomerasepositive; (b) introducing said at least one selected cell to a culturemedium; and (c) proliferating said at least one selected cell in theculture medium.